1. Field of the Invention
This invention relates generally to preparation of blood products for therapeutic use; and more particularly to methods for reducing the infectivity of a virus in human blood-clotting factor products. The method is of particular interest in regard to viruses that cause various forms of hepatitis or that cause acquired immune deficiency syndrome ("AIDS"); and also cytomegalovirus.
2. Prior Art
Isolation of clotting factors in human blood has been indispensable in understanding the pathology of hemophilia and other inherited bleeding disorders. Concomitantly, development of plasma-fractionation schemes for obtaining practical quantities of clotting-factor concentrates has provided important therapeutic tools for such disorders.
Transfusion therapy employing Factor VIII and Factor IX concentrates in particular has proven quite successful in ministering to hemophiliac patients. Under some circumstances, fibrinogen too has therapeutic value. Other clotting factors too are important.
Unfortunately, however, one serious drawback remains associated with transfusion therapy: risk of transmitting hepatitis viruses, AIDS viruses and cytomegalovirus. These viruses include (but are not limited to) hepatitis B virus, the virus known as "hepatitis non-A, non-B virus(es)," and hepatitis delta agent; and the viruses now believed to be causative agents of AIDS, denominated most commonly as HIV, but perhaps also including HIV-2, HIV-3 and HIV-4.
For definiteness of syntax I shall in this document refer to all such AIDS-causing virus or viruses as "AIDS virus." It will be understood, however, that this nomenclature encompasses perhaps more than one virus that may be involved.
Following is a brief description of the mechanisms by which viral transmission can occur in coagulation-factor transfusion therapy. I shall begin with the procedures used for preparing human coagulation-factor products. As is well known, such products usually take the form of human coagulation-factor concentrates, but my invention is not limited to concentrates.
Such concentrates are isolated from human blood plasma, by any of various processes called "plasma fractionation." A typical fractionation scheme is described in Seminars in Thrombosis and Hemostatis, volume VI number 1, page 4 (1979). This process yields cryoprecipitate and supernatant--the former fraction constituting a source of both Factor VIII of Factor IX concentrate in addition to Factor II, VII and X concentrates.
As Gerety and Eyster have demonstrated by their contribution "Hepatitis Among Hemophiliacs," in Non-A, non-B Hepatitis, pages 103 through 106 (1981), hepatitis B virus initially present in whole plasma is distributed to the Factor VIII and Factor IX derivatives during the plasma-fractionation process. As also demonstrated by Maynard and Bradley's paper "Transmission by Blood Products," in the same book at pages 78 and 79, non-A, non-B hepatitis exists in both Factor VIII and IX derivatives.
Thus in simple terms, hepatitis viruses survive the processes used to prepare coagulation-factor concentrates. It is well known that AIDS virus too can be transmitted by Factor VIII and IX concentrates which have not undergone viral inactivation. Cytomegalovirus also may be of concern. Considering hepatitis, AIDS and possibly cytomegalovirus, there is a clear risk of viral infection by coagulation-factor transfusion.
This risk is a serious one. It is serious because a large number of plasma donors is required for commercial "pooled" production of clotting-factor concentrates. In absolute terms, if one donor is infected then possibly the entire supply may be infected, although for various reasons even this can be uncertain.
It is further uncertain to me whether "dilution" of one infected donation in the pool of donations diminishes the risk of infection from that one donation. Even if it does, the quantitative effect is speculative.
Typically the number of donors contributing to a pooled supply does not exceed a few hundred or a few thousand. Hence such dilution in the pool does not normally exceed two or three "logs" (factors of ten). Furthermore, if such a pool has infected donations, then the number of logs of effective dilution may be thereby reduced.
What is generally considered necessary is reduction of viral concentrations by several logs. Thus purity can only be guaranteed by assuring that all the donors are virus free, or by a disinfection procedure that is effective.
Viral transmission by heat-stable plasma components --that is, components other than those related to blood coagulation--an be controlled with comparative ease. Heating the stable components at moderate temperatures and for moderate times suffices to substantially inactivate some of the most troublesome viruses.
For example, hepatitis transmission by albumin can be prevented by heating the albumin in solution at sixty degrees Centigrade for ten hours. Preliminary propositions for similarly inactivating hepatitis viruses in immune globulin by such pasteurization have also been reported -though, to the best of my knowledge, nowhere in the world is a product available from such a procedure, and nowhere have details of how to produce such a product appeared (Welch, A. G. et al., "Non-A, Non-B Hepatitis from Intravenous Immunoglobulin,"
Unfortunately, however, similar efforts to treat clotting-factor concentrates in solution have failed. First, heating of the concentrates alone in solution is believed to markedly reduce or eliminate clotting-factor activity in the concentrates.
Factor VIII concentrate is known to have a very brief half-life even relative to some other coagulation-related factors. The fragility of Factor VIII and its related heat-instability have in the past presented serious impediments to developing virus-free Factor VIII concentrate, even while progress in removing hepatitis virus from other plasma fractions was being made.
Secondly, and more recently, highly purified Factor VIII precipitate has been dissolved in a solution of sucrose glycine and then, while in solution, heated for ten hours at sixty degrees. (All temperatures in this document are expressed in Centigrade degrees.) Although the Factor VIII concentrate subsequently derived from the heated precipitate does retain clotting factor activity, the yields obtained using this approach are very low--e. g., about eight percent (8%).
This work is reported by Heimburger et al. Their papers appear in Hemostasis, volume 10 supplement 1, page 204 (1981); and in the journal Blut, volume 44, pages 249 through 251 (1982).
Therefore, heating in solution does not appear to offer a practical answer to the problem of hepatitis and AIDS transmission by clotting-factor transfusions. The art and science of coagulation therapy, however, has already moved beyond such pasteurization techniques.
In particular a prior patent of the present inventor, U.S. Pat. No. 4,456,590, discloses a method for inactivating viruses present in Factor VIII and IX concentrates. In that process the concentrates are lyophilized (freeze dried), then heated while they remain in the lyophilized state, and eventually reconstituted for therapeutic use.
The lyophilized condition is believed to enhance stability of the concentrates during the heating step. Once lyophilized, the concentrates are heated for very extended periods of time, usually at moderate temperatures.
For example, one major United States manufacturer of clotting-factor concentrates heats the material, while it is lyophilized, at sixty degrees for thirty hours. Another manufacturer heats Factor IX at that temperature for approximately 144 hours. Still another manufacturer heats Factor VIII at sixty-eight degrees; in England, however, the preferred standard for Factor VIII treatment appears to be eighty degrees for seventy-two hours.
Not only the lyophilization-and-heating technique generally, but also these specific time-and-temperature combinations in particular, thus have attained acceptance by some workers in clotting-factor therapy. It must be appreciated that regulatory agencies in the United States and elsewhere continue to exercise very extensive control over techniques.
Any such procedure must perform the function of discriminating between viruses and clotting factors. It must inactivate the viruses to a satisfactory extent, while leaving the activity of the factors intact to a satisfactory extent. Furthermore the resulting clotting-factor product must be safe for human intravenous administration.
Hence a particular combination of parameters must be tested in extensive and costly clinical trials to establish that it satisfies all three of these criteria reliably. Once so tested, a specific procedure cannot be varied without risk of undermining one or another of the three established assurances.
Regulatory agencies may accordingly refuse to allow a manufacturer who heats at sixty degrees for thirty hours to raise the temperature to sixty-four degrees. This is so even though another manufacturer already employs sixty-eight degrees.
Such an intermediate temperature might seem very probably safe and effective; nonetheless, it has not been fully tested. Accordingly the continuation of strict regulatory control is justified.
This continuing control establishes a very definite and restricted set of times and temperatures that are accepted by regulatory agencies as:
(1) adequate for at least some disinfection of lyophilized concentrate, PA1 (2) essentially nondamaging to lyophilized concentrate, and PA1 (3) safe to the product recipient. PA1 (1) lyophilizing the concentrate or obtaining the concentrate prelyophilized; PA1 (2) then heating the concentrate, while it is lyophilized or prelyophilized, for and at a plurality of different time-and-temperature combinations; and PA1 (3) then reconstituting the concentrate. PA1 a much higher assurance of viral inactivation can be obtained for a given expenditure of time and power; or PA1 much less time and power can be expended for the same assurance of viral inactivation; or PA1 a suitable tradeoff can be found that produces significantly higher assurance for significantly less time and power. PA1 (1) for some viruses, "overkill" (that is, reduction of their infectivity far beyond levels at which they pose real threats), while PA1 (2) for some other viruses, at best marginal.
In the remainder of this document--and particularly in the claims which follow this disclosure--all references to "accepted" times or temperatures mean those definite parameter values that have been accepted by regulatory agencies for clinical use.
In adopting this definition of "accepted" time or temperature I do not mean to limit myself to the four time-and-temperature examples that have been stated above. From time to time, manufacturer to manufacturer, and country to country, many other specific values of time or temperature have been or will be so "accepted."
Heat treatment of lyophilized Factor VIII and IX concentrates is in my opinion the most economical and best procedure heretofore available for disinfection against hepatitis and AIDS. In addition it does not introduce any potentially toxic chemicals to inactivate the viruses in the concentrate.
Nevertheless, this procedure like any is subject to improvement. Further quality assurance, if obtained cost-effectively, is always desirable. Moreover, some workers have suggested that inactivation of hepatitis viruses by the method of my prior patent may need some improvement in increasing viral inactivation at some time-and-temperature combinations--particularly those involving relatively short times and low temperatures.
Another area for improvement is that of certain very slight variations in treatment conditions. Laboratory and scrupulously followed. Certain obscure opportunities for marginal performance, however, can creep into even the best procedures.
Some of these variations may be essentially uncontrolled--or even uncontrollable. For example, an incubator, water bath or oven is sometimes used for heat-treating a large number of discrete quantities of clotting-factor concentrate together, at the same time. Despite good design, temperature within a heating system is never perfectly uniform. As the equipment ages, subtle changes can perhaps compound the nonuniformity of temperature.
As a result, those discrete quantities (in individual jars, watchglasses, or otherwise) of clotting-factor concentrate placed in certain parts of the heating system may not reach the same temperature as others. In short, heating conditions are subject to slight variation from quantity to quantity.
If the variation is between, say, one and three degrees, treatment of those particular quantities of clotting factor might be considered marginal. Therefore, to provide a natural opportunity for minimizing the effects of such uncontrolled variations would be desirable.
Certain information that appears in the literature will be introduced here. I refer to a paper by Piszkiewicz et al., "Heat Inactivation of Human Immunodeficiency Virus in Lyophilized Anti-Inhibitor Coagulant Complex (*Autoplex(R)*)," in Thrombosis Research volume 44, pages 701 through 707 (1986)
The Piszkiewicz article, and earlier papers to which it refers, show that viral inactivation does not follow a simple exponential function. In other words, inactivation does not continue at a constant rate, or fractional reduction, relative to the infectivity at each time.
Rather, as the treatment proceeds the inactivation decelerates (even as compared with exponential decay). Most of the viral inactivation occurs in initial periods of heating, and particularly in the first hours. Piszkiewicz fails to suggest any way to use these observations or conclusions to improve disinfection techniques.